NK cells possess several sets of activating and inhibitory receptors that control different steps in cytotoxic lymphocyte-mediated killing of target cells, including conjugation of NK cells to target cells, polarization of lytic granules towards target cells, and degranulation. Prior engagement of NK cells with MHC class I molecules by inhibitory receptors allows for greater intrinsic responsiveness to subsequent activation stimuli through a process called NK cell licensing (aka education). The number of inhibitory receptors engaged with MHC and the strength of MHC binding to inhibitory receptors calibrate the potential responsiveness of each NK cell for cytotoxicity and cytokine secretion. We tested whether the weaker inhibitory receptor KIR2DL3, which binds HLA-C group 1 alleles, conferred weaker licensing than the stronger KIR2DL1, which binds HLA-C group 2 alleles. We analyzed degranulation in NK cell subsets expressing single and multiple receptors for HLA class I. NKG2A, which binds HLA-E, had the strongest licensing impact, while KIR2DL3, KIR2DL1, and KIR3DL1, which binds HLA-B, were weaker but similar to each other. Thus, KIR2DL3 and KIR2DL1 have similar capacity to license NK cells, suggesting that inhibitory signal strength and amount of available HLA-C ligands do not correlate with NKC licensing. Therefore, the basis for disease associations with specific combinations of HLA-C and KIR2DL likely encompasses factors other than licensing. KIR2DL1, KIR2DL2 and KIR2DL3 bind dimorphic forms of HLA-C alleles defined as group C1 and C2. KIR2DL2 and KIR2DL3 bind group C1 alleles that contain Ser77 and Asn80 while KIR2DL1 binds group C2 alleles that contain Asn77 and Lys80. However, this binary system has been challenged as some HLA-C2 alleles showed binding to KIR2DL2 and KIR2DL3. In particular, the group C2 allele HLA-C*05:01 bound KIR2DL1, KIR2DL2 and KIR2DL3 with equally high intensity. As it is known that KIR binding to HLA-C is peptide selective, we hypothesized that peptides bound to HLA-C*05:01 may confer binding to KIR2DL1, KIR2DL2 and KIR2DL3. To this end, we eluted and sequenced peptides bound to HLA-C*05:01. We generated TAP deficient HLA-C*05:01 cells through expression of the viral TAP inhibitor ICP47. We monitored the capacity of individual synthetic HLA-C*05:01 peptides to confer direct binding of soluble KIR2DL1, KIR2DL2 and KIR2DL3 molecules, and functional inhibition of KIR2DL1+ and KIR2DL3+ NK cells. We showed that of twenty peptides tested, 16 peptides conferred binding to KIR2DL1 when presented on .221-C*05:01-ICP47, while only two conferred binding to KIR2DL2 and KIR2DL3. The capacity to bind KIR2DL2 and KIR2DL3 was dependent on peptide side chains at positions 7 and 8 and was competent for inhibition of KIR2DL3+ NK cells. These two peptides were also competent for KIR2DL1 binding and inhibition of KIR2DL1+ NK cells showing that these two peptides can override KIR specificity defined by group C1 and C2. Signaling by immunoreceptors is initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within immunoreceptor tyrosine-based inhibition motifs (ITIM) results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work has shown that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. We have shown that substitution of histidine 36 (His-36) in the first Ig domain of KIR2DL1 with alanine (H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, and recruitment of tyrosine phosphatase SHP-1. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases, and that KIR binding to HLA-C must override the hindrance His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C+ target cells than expression of wild type KIR2DL1. These results have revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1. IL-15 is essential for NK cell proliferation, activation and survival. IL-15 bound to the IL-15 receptor alpha chain (IL-15Ralpha) is presented in trans to cells bearing the IL-2 receptor beta and common gamma chains. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptorligand interactions. We tested human NK cells for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Ralpha. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIRHLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to down-regulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A+ cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Co-engagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 kinase. These results demonstrate a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.